Our overall goal is to understand the signaling pathways used by interferons (IFNs), from receptors to genes. The IFNs regulate antiviral responses, cell growth, and immune responses, and it is worthwhile per se to study how they accomplish these diverse purposes. We already understand IFN transcriptional signaling pathways better than those used by growth factors and other cytokines. Since there are important similarities among many signaling pathways, it is likely that improved knowledge of the IFN pathways will help to advance our understanding of other pathways as well, such as those for IL3, erythropoietin, growth hormone, EGF and PDGF. We have generated 12 mutant cell lines defective in response to IFNs and have used them to provide important new information about signaling. The mutants fall into several groups: unresponsive to IFN-alpha, responsive IFN-beta and IFN-gamma, unresponsive to IFNs -alpha and -beta, responsive to IFN-gamma, unresponsive to IFNs -alpha, -beta, and -gamma, unresponsive to IFN-gamma only (all genes); unresponsive to IFN-gamma only (only class II and invariant chain genes). All the mutants are recessive and stable and thus can be used to clone the affected genes. Complementation has led to identification of three essential protein tyrosine kinases. tyk2 is essential for response to IFN-alpha, JAK2 is essential for response to IFN-gamma, JAK1 is essential for response to all type I and type II IFNs. Two other mutants have been complemented by cDNAs that encode the p48 and p91 subunits of the key transcription factor ISGF3. The p91 subunit is also essential for response to all type I and type II IFNs. There are three main aims. (1) We will isolate new mutants by using approaches that have already been successful, using mutagenesis with ICR- 191, followed by lethal selection or selection for failure of IFN-alpha inducible proteins to appear on the cell surface. We will then complement these mutants with cDNAs or genomic DNAs to identify and clone the missing component. (2) We will obtain new types of mutants by cloning dominant genetic suppressor elements (GSEs) from large cDNA libraries. (3) We will explore the interrelationship of the components of the putative "supercomplex" of proteins associated with the cytoplasmic face of the IFN-alpha receptor, which may include the known tyrosine kinases tyk2 and JAK1, the known transcription factor elements p91 and p113, and as yet unknown components such as protein tyrosine phosphatases and anchor components. Protein-protein crosslinking will be a major tool.